The long term goal of our research is to understand how proteins fold in living cells. Chaperonins are key components of the cellular folding machinery. These large protein complexes, consisting of two stacked seven- to nine-membered rings, bind unfolded substrates in their central cavity and use binding and hydrolysis of ATP to mediate polypeptide folding. The folding of substrate proteins occurs in the central cavity formed by each ring. There are substantial differences between group I chaperonins, found in prokaryotic cells, and the distantly related group II chaperonins in Archaea and Eukarya,. Group I chaperonins require a ring-shaped cofactor, such GroES for GroEL, that upon binding acts as a lid for the cavity, creating a folding chamber that encloses polypeptide substrates. Group II chaperonins are heterooligomeric and lack a GroES-like cofactor, suggesting that their conformational cycle is significantly different from group I chaperonins. The present proposal aims to elucidate the mechanism of the eukaryotic chaperonin TRiC (TCP1-Ring Complex, also called CCT). Despite its essential role in polypeptide folding, little is known about the mechanism and substrate binding properties of TRiC. To understand how TRiC facilitates folding we propose the following specific aims: 1. Characterize of the nucleotide cycle of the chaperonin TRiC; 2. Define the molecular basis of TRiC-substrate interactions; 3. Explore the mechanism of TRiC-assisted folding.